This proposal is focused on the role of gap junctions in retinal circuitry, and specifically, on the mechanism by which rod input switches to cone input during light adaptation. Gap junctions play a critical role in the retina and they occur frequently between many cell types. In horizontal cells, gap junctions regulate the size of the receptive field and thereby control the strength of feedback to photoreceptors. In the mammalian retina, rod signals enter cone pathways via gap junctions between the AII or rod amacrine cell and cone bipolar cells. Gap junctions have been observed morphologically and inferred from studies of dye coupling. The correlation between dye coupling and gap junctions is strong with several examples of the EM visualization of gap junctions in dye coupled neurons. Specific Aim 1. The PI has identified a series of 6 biotinylated tracers, with molecular weights from 286 to 555, that can be used as probes for gap junctions, to compare the relative permeability of gap junctions from different cell types and thus determine if different cells synthesize different connexons. One dye, biotin-x-cadaverine, MW 442, passes through gap junctions that exclude Lucifer Yellow, MW 443. This suggests that Lucifer Yellow is excluded by charge instead of size. Preliminary evidence suggests that intermediate members of the series will provide improved staining and some of the higher members of the series may be selective for certain types of gap junction. Specific Aim 2. The PI has identified a calbindin labeled cone bipolar cell which is extensively coupled to AII amacrine cells and, by double label EM, recovered the gap junctions that are the morphological substrate for dye coupling between these cells. The PI will build on these results, and by a combination of intracellular dye injection and confocal microscopy, determine whether the calbindin bipolar cells contact ON alpha ganglion cells. Synaptic contacts will be confirmed by EM. This will complete the last step in the rod pathway: starting with the rod and progress to the rod bipolar cell to AII to gap junction to calbindin bipolar cell and finally to the alpha ganglion cell. Preliminary data, using the probes described above, suggest that the AII/calbindin bipolar gap junction are modulated by NO/cGMP. Specific Aim 3. The gap junctions between AII amacrine cells and cone bipolar cells are a key site for regulation of rod vs cone input to ganglion cells. The PI has shown that NOS is present in a dense matrix at this level, that NO stimulated cGMP is localized to ON cone bipolar cells and that AII/bipolar cell dye coupling is modulated by NO/cGMP, as opposed to dopamine. The PI will test the effect of NO on the dark adapted input to brisk transient ON ganglion cells, the physiological correlate of a ganglion cells. This signal must pass through the AII/calbindin bipolar gap junctions described above. This will determine if NO is the signal to close the AII/bipolar gap junctions in the light, thereby closing the shunt to AII amacrine cells and excluding the slow, noisy rod signals from the faster, high acuity cone pathway.